Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an attenuated acek gene

ABSTRACT

The invention relates to a process for the preparation of L-amino acids, in particular L-threonine, in which the following steps are carried out: a) fermentation of microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which the aceK gene, or the nucleotide sequence which codes for this, is attenuated, in particular eliminated, b) concentration of the L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the L-amino acid.

FIELD OF THE INVENTION

This invention relates to a process for the fermentative preparation ofL-amino acids, in particular L-threonine, using strains of theEnterobacteriaceae family in which the aceK gene is attenuated.

PRIOR ART

L-Amino acids, in particular L-threonine, are used in human medicine andin the pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

It is known to prepare L-amino acids by fermentation of strains ofEnterobacteriaceae, in particular Escherichia coli (E. coli) andSerratia marcescens. Because of their great importance, work isconstantly being undertaken to improve the preparation processes.Improvements to the process can relate to fermentation measures, such ase.g. stirring and supply of oxygen, or the composition of the nutrientmedia, such as e.g. the sugar concentration during the fermentation, orthe working up to the product form, by e.g. ion exchange chromatography,or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites, such as e.g. the threonine analogueα-amino-β-hydroxyvaleric acid (AHV), or are auxotrophic for metabolitesof regulatory importance and produce L-amino acid, such as e.g.L-threonine, are obtained in this manner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of strains of the Enterobacteriaceaefamily which produce L-amino acids, by amplifying individual amino acidbiosynthesis genes and investigating the effect on the production.

OBJECT OF THE INVENTION

The object of the invention is to provide new measures for improvedfermentative preparation of L-amino acids, in particular L-threonine.

SUMMARY OF THE INVENTION

The invention provides a process for the fermentative preparation ofL-amino acids, in particular L-threonine, using microorganisms of theEnterobacteriaceae family which in particular already produce L-aminoacids and in which the nucleotide sequence which codes for the aceK geneis attenuated.

DETAILED DESCRIPTION OF THE INVENTION

Where L-amino acids or amino acids are mentioned in the following, thismeans one or more amino acids, including their salts, chosen from thegroup consisting of L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan and L-arginine. L-Threonine is particularly preferred.

The term “attenuation” in this connection describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or a gene or allele which codes fora corresponding enzyme with a low activity or inactivates thecorresponding enzyme (protein) or gene, and optionally combining thesemeasures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

The process comprises carrying out the following steps:

-   -   a) fermentation of microorganisms of the Enterobacteriaceae        family in which the aceK gene is attenuated,    -   b) concentration of the corresponding L-amino acid in the medium        or in the cells of the microorganisms of the Enterobacteriaceae        family, and    -   c) isolation of the desired L-amino acid, constituents of the        fermentation broth and/or the biomass in its entirety or        portions (>0 to 100%) thereof optionally remaining in the        product.

The microorganisms which the present invention provides can produceL-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, optionally starch, optionally cellulose or from glycerol andethanol. They are representatives of the Enterobacteriaceae familychosen from the genera Escherichia, Erwinia, Providencia and Serratia.The genera Escherichia and Serratia are preferred. Of the genusEscherichia the species Escherichia coli and of the genus Serratia thespecies Serratia marcescens are to be mentioned in particular.

Suitable strains, which produce L-threonine in particular, of the genusEscherichia, in particular of the species Escherichia coli, are, forexample

-   Escherichia coli TF427-   Escherichia coli H4578-   Escherichia coli KY10935-   Escherichia coli VNIIgenetika MG442-   Escherichia coli VNIIgenetika M1-   Escherichia coli VNIIgenetika 472T23-   Escherichia coli BKIIM B-3996-   Escherichia coli kat 13-   Escherichia coli KCCM-10132

Suitable L-threonine-producing strains of the genus Serratia, inparticular of the species Serratia marcescens, are, for example

-   Serratia marcescens HNr21-   Serratia marcescens TLr156-   Serratia marcescens T2000

Strains from the Enterobacteriaceae family which produce L-threoninepreferably have, inter alia, one or more genetic or phenotypic featureschosen from the group consisting of: resistance toα-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance toethionine, resistance to α-methylserine, resistance to diaminosuccinicacid, resistance to α-aminobutyric acid, resistance to borrelidin,resistance to rifampicin, resistance to valine analogues, such as, forexample, valine hydroxamate, resistance to purine analogues, such as,for example, 6-dimethylaminopurine, a need for L-methionine, optionallya partial and compensable need for L-isoleucine, a need formeso-diaminopimelic acid, auxotrophy in respect of threonine-containingdipeptides, resistance to L-threonine, resistance to L-homoserine,resistance to L-lysine, resistance to L-methionine, resistance toL-glutamic acid, resistance to L-aspartate, resistance to L-leucine,resistance to L-phenylalanine, resistance to L-serine, resistance toL-cysteine, resistance to L-valine, sensitivity to fluoropyruvate,defective threonine dehydrogenase, optionally an ability for sucroseutilization, enhancement of the threonine operon, enhancement ofhomoserine dehydrogenase I-aspartate kinase I, preferably of the feedback resistant form, enhancement of homoserine kinase, enhancement ofthreonine synthase, enhancement of aspartate kinase, optionally of thefeed back resistant form, enhancement of aspartate semialdehydedehydrogenase, enhancement of phosphoenol pyruvate carboxylase,optionally of the feed back resistant form, enhancement of phosphoenolpyruvate synthase, enhancement of transhydrogenase, enhancement of theRhtB gene product, enhancement of the RhtC gene product, enhancement ofthe YfiK gene product, enhancement of a pyruvate carboxylase, andattenuation of acetic acid formation.

It has been found that microorganisms of the Enterobacteriaceae familyproduce L-amino acids, in particular L-threonine, in an improved mannerafter attenuation, in particular elimination, of the aceK gene.

The nucleotide sequences of the genes of Escherichia coli belong to theprior art and can also be found in the genome sequence of Escherichiacoli published by Blattner et al. (Science 277: 1453-1462 (1997)).

The aceK gene is described, inter alia, by the following data:

-   Description: Isocitrate dehydrogenase kinase/phosphatase-   EC No.:-   2.7.1.116    -   3.1.3.—-   Reference: Cortay et al.; Journal of Bacteriology 170(1): 89-97    (1988); Klumpp et al.; Journal of Bacteriology 170(6): 2763-2769    (1988); Nelson et al.; Genetics 147(4): 1509-1520 (1997); Laporte et    al.; Biochimie 71(9-10): 1051-7 (1989)-   Accession No.: AE000474

The nucleic acid sequences can be found in the databanks of the NationalCenter for Biotechnology Information (NCBI) of the National Library ofMedicine (Bethesda, Md., USA), the nucleotide sequence databank of theEuropean Molecular Biologies Laboratories (EMBL, Heidelberg, Germany orCambridge, UK) or the DNA databank of Japan (DDBJ, Mishima, Japan).

The genes described in the text references mentioned can be usedaccording to the invention. Alleles of the genes which result from thedegeneracy of the genetic code or due to “sense mutations” of neutralfunction can furthermore be used.

To achieve an attenuation, for example, expression of the gene or thecatalytic properties of the enzyme proteins can be reduced oreliminated. The two measures can optionally be combined.

The reduction in gene expression can take place by suitable culturing,by genetic modification (mutation) of the signal structures of geneexpression or also by the antisense-RNA technique. Signal structures ofgene expression are, for example, repressor genes, activator genes,operators, promoters, attenuators, ribosome binding sites, the startcodon and terminators. The expert can find information in this respect,inter alia, for example, in Jensen and Hammer (Biotechnology andBioengineering 58: 191-195 (1998)), in Carrier and Keasling(Biotechnology Progress 15: 58-64 (1999), Franch and Gerdes (CurrentOpinion in Microbiology 3: 159-164 (2000)) and in known textbooks ofgenetics and molecular biology, such as, for example, the textbook ofKnippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, GeorgThieme Verlag, Stuttgart, Germany, 1995) or that of Winnacker (“Gene undKlone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany,1990).

Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art. Exampleswhich may be mentioned are the works of Qiu and Goodman (journal ofBiological Chemistry 272: 8611-8617 (1997)), Yano et al. (Proceedings ofthe National Academy of Sciences, USA 95: 5511-5515 (1998)), Wente andSchachmann (Journal of Biological Chemistry 266: 20833-20839 (1991)).Summarizing descriptions can be found in known textbooks of genetics andmolecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik[General Genetics]”, Gustav Fischer verlag, Stuttgart, 1986).

Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair in a genelead to “frame shift mutations”, which lead to incorrect amino acidsbeing incorporated or translation being interrupted prematurely. If astop codon is formed in the coding region as a consequence of themutation, this also leads to a premature termination of the translation.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik[Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”,VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, 1986).

Suitable mutations in the genes, such as, for example, deletionmutations, can be incorporated into suitable strains by gene or allelereplacement.

A conventional method is the method, described by Hamilton et al.(Journal of Bacteriology 171: 4617-4622 (1989)), of gene replacementwith the aid of a conditionally replicating pSC101 derivative pMAK705.Other methods described in the prior art, such as, for example, those ofMartinez-Morales et al. (Journal of Bacteriology 181: 7143-7148 (1999))or those of Boyd et al. (Journal of Bacteriology 182: 842-847 (2000)),can likewise be used.

It is also possible to transfer mutations in the particular genes ormutations which affect expression of the particular genes into variousstrains by conjugation or transduction.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, with strains of the Enterobacteriaceaefamily, in addition to attenuation of the aceK gene, for one or moreenzymes of the known threonine biosynthesis pathway or enzymes ofanaplerotic metabolism or enzymes for the production of reducednicotinamide adenine dinucleotide phosphate or enzymes of glycolysis orPTS enzymes or enzymes of sulfur metabolism to be enhanced.

The term “enhancement”¹ in this connection describes the increase in theintracellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or a gene which codes for a corresponding enzyme or proteinwith a high activity, and optionally combining these measures.

By enhancement measures, in particular over-expression, the activity orconcentration of the corresponding protein is in general increased by atleast 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to amaximum of 1000% or 2000%, based on that of the wild-type protein or theactivity or concentration of the protein in the starting microorganism.

Thus, for example, at the same time one or more of the genes chosen fromthe group consisting of

-   -   the thrABC operon which codes for aspartate kinase, homoserine        dehydrogenase, homoserine kinase and threonine synthase (U.S.        Pat. No. 4,278,765),    -   the pyc gene of Corynebacterium glutamicum which codes for        pyruvate carboxylase (WO 99/18228),    -   the pps gene which codes for phosphoenol pyruvate synthase        (Molecular and General Genetics 231(2): 332-336 (1992)),    -   the ppc gene which codes for phosphoenol pyruvate carboxylase        (Gene 31: 279-283 (1984)),    -   the pntA and pntB genes which code for transhydrogenase        (European Journal of Biochemistry 158: 647-653 (1986)),    -   the rhtB gene which imparts homoserine resistance (EP-A-0 994        190),    -   the mqo gene which codes for malate:quinone oxidoreductase (WO        02/06459),    -   the rhtC gene which imparts threonine resistance (EP-A-1 013        765),    -   the thrE gene of Corynebacterium glutamicum which codes for the        threonine export protein (WO 01/92545),    -   the gdhA gene which codes for glutamate dehydrogenase (Nucleic        Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983)),    -   the hns gene which codes for the DNA-binding protein HLP-II        (Molecular and General Genetics 212: 199-202 (1988)),    -   the pgm gene which codes for phosphoglucomutase (Journal of        Bacteriology 176: 5847-5851 (1994)),    -   the fba gene which codes for fructose biphosphate aldolase        (Biochemical Journal 257: 529-534 (1989)),    -   the ptsH gene of the ptsHIcrr operon which codes for the        phosphohistidine protein hexose phosphotransferase of the        phosphotransferase system PTS (Journal of Biological Chemistry        262: 16241-16253 (1987)),    -   the ptsI gene of the ptsHIcrr-operon which codes for enzyme I of        the phosphotransferase system PTS (Journal of Biological        Chemistry 262: 16241-16253 (1987)),    -   the crr gene of the ptsHIcrr operon which codes for the        glucose-specific IIA component of the phosphotransferase system        PTS (Journal of Biological Chemistry 262: 16241-16253 (1987)),    -   the ptsG gene which codes for the glucose-specific IIBC        component (journal of Biological Chemistry 261: 16398-16403        (1986)),    -   the lrp gene which codes for the regulator of the leucine        regulon (Journal of Biological Chemistry 266: 10768-10774        (1991)),    -   the mopB gene which codes for 10 Kd chaperone (Journal of        Biological Chemistry 261: 12414-12419 (1986)) and is also known        by the name groES,    -   the ahpC gene of the ahpCF operon which codes for the small        sub-unit of alkyl hydroperoxide reductase (Proceedings of the        National Academy of Sciences of the United States of America 92:        7617-7621 (1995)),    -   the ahpF gene of the ahpCF operon which codes for the large        sub-unit of alkyl hydroperoxide reductase (Proceedings of the        National Academy of Sciences USA 92: 7617-7621 (1995)),    -   the cysK gene which codes for cysteine synthase A (Journal of        Bacteriology 170: 3150-3157 (1988)),    -   the cysB gene which codes for the regulator of the cys regulon        (Journal of Biological Chemistry 262: 5999-6005 (1987)),    -   the cysJ gene of the cysJIH operon which codes for the        flavoprotein of NADPH sulfite-reductase (Journal of Biological        Chemistry 264: 15796-15808 (1989), Journal of Biological        Chemistry 264: 15726-15737 (1989)),    -   the cysI gene of the cysJIH operon which codes for the        haemoprotein of NADPH sulfite reductase (Journal of Biological        Chemistry 264: 15796-15808 (1989), Journal of Biological        Chemistry 264: 15726-15737 (1989)) and    -   the cysH gene of the cysJIH operon which codes for adenylyl        sulfate reductase (Journal of Biological Chemistry 264:        15796-15808 (1989), Journal of Biological Chemistry 264:        15726-15737 (1989))        can be enhanced, in particular over-expressed.

The use of endogenous genes is in general preferred. “Endogenous genes”or “endogenous nucleotide sequences” are understood as meaning the genesor nucleotide sequences present in the population of a species.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, in addition to attenuation of the aceK gene,for one or more of the genes chosen from the group consisting of

-   -   the tdh gene which codes for threonine dehydrogenase (Journal of        Bacteriology 169: 4716-4721 (1987)),    -   the mdh gene which codes for malate dehydrogenase (E.C.        1.1.1.37) (Archives in Microbiology 149: 36-42 (1987)),    -   the gene product of the open reading frame (orf) yjfA (Accession        Number AAC77180 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA)),    -   the gene product of the open reading frame (orf) ytfP (Accession        Number AAC77179 of the National Center for Biotechnology        Information (NCBI, Bethesda, Md., USA)),    -   the pckA gene which codes for the enzyme phosphoenol pyruvate        carboxykinase (Journal of Bacteriology 172: 7151-7156 (1990)),    -   the poxB gene which codes for pyruvate oxidase (Nucleic Acids        Research 14(13): 5449-5460 (1986)),    -   the aceA gene which codes for the enzyme isocitrate lyase        (Journal of Bacteriology 170: 4528-4536 (1988)),    -   the dgsA gene which codes for the DgsA regulator of the        phosphotransferase system (Bioscience, Biotechnology and        Biochemistry 59: 256-261 (1995)) and is also known under the        name of the mlc gene,    -   the fruR gene which codes for the fructose repressor (Molecular        and General Genetics 226: 332-336 (1991)) and is also known        under the name of the cra gene,    -   the rpoS gene which codes for the sigma³⁸ factor (WO 01/05939)        and is also known under the name of the katF gene,    -   the aspA gene which codes for aspartate ammonium lyase (Nucleic        Acids Research 13(6): 2063-2074 (1985)),    -   the aceB gene which codes for malate synthase A (Nucleic Acids        Research 16(19): 9342 (1988) and    -   the ugpB gene which codes for the periplasmic binding protein of        the sn-glycerol 3-phosphate transport system (Molecular        Microbiology 2(6): 767-775 (1988))        to be attenuated, in particular eliminated or for the expression        thereof to be reduced.

It may furthermore be advantageous for the production of L-amino acids,in particular L-threonine, in addition to attenuation of the aceK gene,to eliminate undesirable side reactions (Nakayama: “Breeding of AminoAcid Producing Microorganisms”, in: Overproduction of MicrobialProducts, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,1982).

The microorganisms produced according to the invention can be culturedin the batch process (batch culture), the fed batch process (feedprocess) or the repeated fed batch process (repetitive feed process). Asummary of known culture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktorenund periphere Einrichtungen [Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and optionally cellulose, oils andfats, such as e.g. soya oil, sunflower oil, groundnut oil and coconutfat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleicacid, alcohols, such as e.g. glycerol and ethanol, and organic acids,such as e.g. acetic acid, can be used as the source of carbon. Thesesubstances can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as e.g. magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances, such asamino acids and vitamins, can be employed in addition to theabovementioned substances. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as e.g. fatty acid polyglycol esters, canbe employed to control the development of foam. Suitable substanceshaving a selective action, e.g. antibiotics, can be added to the mediumto maintain the stability of plasmids. To maintain aerobic conditions,oxygen or oxygen-containing gas mixtures, such as e.g. air, areintroduced into the culture. The temperature of the culture is usually25° C. to 45° C., and preferably 30° C. to 40° C. Culturing is continueduntil a maximum of L-amino acids or L-threonine has formed. This targetis usually reached within 10 hours to 160 hours.

The analysis of L-amino acids can be carried out by anion exchangechromatography with subsequent ninhydrin derivation, as described bySpackman et al. (Analytical Chemistry 30: 1190-1206 (1958)), or it cantake place by reversed phase HPLC as described by Lindroth et al.(Analytical Chemistry 51:. 1167-1174 (1979)).

The process according to the invention is used for the fermentativepreparation of L-amino acids, such as, for example, L-threonine,L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine, inparticular L-threonine.

A pure culture of the Escherichia coli K-12 strain DH5α/pMAK705 wasdeposited as DSM 13720 on 8 Sep. 2000 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques ofrestriction, ligation, Klenow and alkaline phosphatase treatment arecarried out by the method of Sambrook et al. (Molecular Cloning—ALaboratory Manual (1989) Cold Spring Harbor Laboratory Press). Unlessdescribed otherwise, the transformation of Escherichia coli is carriedout by the method of Chung et al. (Proceedings of the National Academyof Sciences of the United States of America, USA 86: 2172-2175 (1989)).

The incubation temperature for the preparation of strains andtransformants is 37° C. Temperatures of 30° C. and 44° C. are used inthe gene replacement method of Hamilton et al.

EXAMPLE 1

Construction of the Deletion Mutation of the aceK Gene

Parts of the gene regions lying upstream and downstream of the aceK geneand parts of the 5′ and 3′ region of the aceK gene are amplified fromEscherichia coli K12 using the polymerase chain reaction (PCR) andsynthetic oligonucleotides. Starting from the nucleotide sequence of theaceK gene and sequences lying upstream and downstream in E. coli K12MG1655 (SEQ ID No. 1, Accession Number AE000474), the following PCRprimers are synthesized (MWG Biotech, Ebersberg, Germany): aceK5′-1:5′ - CAACAACAACCGTTGCTGAC - 3′ (SEQ ID No. 3) aceK5′-2: 5′ -CAGTTCGTTCGCCACCTGTA - 3′ (SEQ ID No. 4) aceK3′-1: 5′ -TGTTCCGCGCTGATTACTGG - 3′ (SEQ ID No. 5) aceK3′-2: 5′ -GAATGGTGATGCCGACATGC - 3′ (SEQ ID No. 6)

The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolatedaccording to the manufacturer's instructions with “Qiagen Genomic-tips100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx. 800 bp in sizefrom the 5′ region of the aceK gene region (called aceK5′) and a DNAfragment approx. 800 bp in size from the 3′ region of the aceK generegion (called aceK3′) can be amplified with the specific primers understandard PCR conditions (Innis et al. (1990) PCR Protocols. A Guide toMethods and Applications, Academic Press) with Taq-DNA polymerase(Gibco-BRL, Eggenstein, Germany). The PCR products are each ligated withthe vector pCR2.1-TOPO (TOPO TA Cloning Kit, Invitrogen, Groningen, TheNetherlands) in accordance with the manufacturer's instructions andtransformed into the E. coli strain TOP10F′. Selection ofplasmid-carrying cells takes place on LB agar, to which 50 μg/mlampicillin are added. After isolation of the plasmid DNA, the vectorpCR2.1-TOPOaceK3′ is cleaved with the restriction enzymes XbaI andEcl136II. The aceK3′ fragment is isolated after separation in 0.8%agarose gel with the aid of the QIAquick Gel Extraction Kit (QIAGEN,Hilden, Germany). After isolation of the plasmid DNA the vectorpCR2.1-TOPOaceK5′ is cleaved with the enzymes EcoRV and XbaI and ligatedwith the aceK3′ fragment isolated. The E. coli strain DH5α istransformed with the ligation batch and plasmid-carrying cells areselected on LB agar, to which 50 μg/ml ampicillin are added. Afterisolation of the plasmid DNA those plasmids in which the mutagenic DNAsequence shown in SEQ ID No. 7 is cloned are detected by controlcleavage with the enzymes BamHI, SpeI and PvuI. One of the plasmids iscalled pCR2.1-TOPOΔaceK (=pCR2.1-TOPOdeltaaceK).

EXAMPLE 2

Construction of the Replacement Vector pBAK705ΔaceK

The ΔaceK allele described in example 1 is isolated from the vectorpCR2.1-TOPOΔaceK after restriction with the enzymes KpnI and XbaI andseparation in 0.8% agarose gel, and ligated with the plasmid pMAK705(Hamilton et al., Journal of Bacteriology 171: 4617-4622 (1989)), whichhas been digested with the enzymes KpnI and XbaI. The ligation batch istransformed in DH5α and plasmid-carrying cells are selected on LB agar,to which 20 μg/ml chloramphenicol are added. Successful cloning isdemonstrated after isolation of the plasmid DNA and cleavage with theenzymes BamHI, MluI and PauI. The replacement vector formed,pMAK705ΔaceK (=pMAK705deltaaceK), is shown in FIG. 1.

EXAMPLE 3

Position-Specific Mutagenesis of the aceK Gene in the E. coli StrainMG442

The L-threonine-producing E. coli strain MG442 is described in thepatent specification U.S. Pat. No. 4,278,765 and deposited as CMIMB-1628 at the Russian National Collection for Industrial Microorganisms(VKPM, Moscow, Russia).

For replacement of the chromosomal aceK gene with the plasmid-codeddeletion construct, MG442 is transformed with the plasmid pMAK705ΔaceK.The gene replacement is carried out using the selection method describedby Hamilton et al. (Journal of Bacteriology 171: 4617-4622 (1989)) andis verified by standard PCR methods (Innis et al. (1990) PCR Protocols.A Guide to Methods and Applications, Academic Press) with the followingoligonucleotide primers: aceK5′-1: 5′ - CAACAACAACCGTTGCTGAC - 3′ (SEQID No. 3) aceK3′-2: 5′ - GAATGGTGATGCCGACATGC - 3′ (SEQ ID No. 6)

After replacement has taken place, MG442 contains the form of the ΔaceKallele shown in SEQ ID No. 8. The strain obtained is called MG442ΔaceK.

EXAMPLE 4

Preparation of L-Threonine with the Strain MG442ΔaceK

MG442ΔaceK is multiplied on minimal medium with the followingcomposition: 3.5 g/l Na₂HPO₄.2H₂₀, 1.5 g/l KH₂PO₄, 1 g/l NH₄Cl, 0.1 g/lMgSO₄.7H₂O, 2 g/l glucose, 20 g/l agar. The formation of L-threonine ischecked in batch cultures of 10 ml contained in 100 ml conical flasks.For this, 10 ml of preculture medium of the following composition: 2 g/lyeast extract, 10 g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5 g/l MgSO₄.7H₂O, 15g/l CaCO₃, 20 g/l glucose are inoculated and the batch is incubated for16 hours at 37° C. and 180 rpm on an ESR incubator from Kühner AG(Birsfelden, Switzerland). 250 μl of this preculture are transinoculatedinto 10 ml of production medium (25 g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/lMgSO₄.7H₂O, 0.03 g/l FeSO₄*7H₂O, 0.018 g/l MnSO₄*1H₂O, 30 g/l CaCO₃, 20g/l glucose) and the batch is incubated for 48 hours at 37° C. After theincubation the optical density (OD) of the culture suspension isdetermined with an LP2W photometer from Dr. Lange (Düsseldorf, Germany)at a measurement wavelength of 660 nm.

The concentration of L-threonine formed is then determined in thesterile-filtered culture supernatant with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column reaction with ninhydrin detection.

The result of the experiment is shown in Table 1. TABLE 1 OD L-ThreonineStrain (660 nm) g/l MG442 6.0 1.5 MG442ΔaceK 5.8 1.7

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: pMAK705ΔaceK (=pMAK705deltaaceK)

The length data are to be understood as approx. data. The abbreviationsand designations used have the following meaning:

-   -   cat: Chloramphenicol resistance gene    -   rep-ts: Temperature-sensitive replication region of the plasmid        pSC101    -   aceK5′: Part of the 5′ region of the aceK gene and the region        lying upstream    -   aceK3′: Part of the 3′ region of the aceK gene and the region        lying downstream

The abbreviations for the restriction enzymes have the following meaning

-   -   BamHI: Restriction endonuclease from Bacillus amyloliquefaciens        H    -   KpnI: Restriction endonuclease from Klebsiella pneumoniae    -   MluI: Restriction endonuclease from Micrococcus luteus IFO 12992    -   PauI: Restriction endonuclease from Paracoccus alcaliphilus    -   XbaI: Restriction endonuclease from Xanthomonas badrii

1. A process for the preparation of L-amino acids, in particularL-threonine, which comprises carrying out the following steps: a)fermentation of microorganisms of the Enterobacteriaceae family whichproduce the desired L-amino acid and in which the aceK gene, or thenucleotide sequences which code for this, is or are attenuated, inparticular eliminated, b) concentration of the desired L-amino acid inthe medium or in the cells of the microorganisms, and c) isolation ofthe desired L-amino acid, constituents of the fermentation broth and/orthe biomass in its entirety or portions (>0 to 100%) thereof optionallyremaining in the product.
 2. A process as claimed in claim 1, whereinmicroorganisms in which further genes of the biosynthesis pathway of thedesired L-amino acid are additionally enhanced are employed.
 3. Aprocess as claimed in claim 1, wherein microorganisms in which themetabolic pathways which reduce the formation of the desired L-aminoacid are at least partly eliminated are employed.
 4. A process asclaimed in claim 1, wherein the expression of the polynucleotide whichcodes for the aceK gene is attenuated, in particular eliminated.
 5. Aprocess as claimed in claim 1, wherein the regulatory and/or catalyticproperties of the polypeptide (enzyme protein) for which thepolynucleotide aceK codes are reduced.
 6. A process as claimed in claim1, wherein, for the preparation of L-amino acids, microorganisms of theEnterobacteriaceae family in which in addition at the same time one ormore of the genes chosen from the group consisting of: 6.1 the thrABCoperon which codes for aspartate kinase, homoserine dehydrogenase,homoserine kinase and threonine synthase, 6.2 the pyc gene which codesfor pyruvate carboxylase, 6.3 the pps gene which codes for phosphoenolpyruvate synthase, 6.4 the ppc gene which codes for phosphoenol pyruvatecarboxylase, 6.5 the pntA and pntB genes which code fortranshydrogenase, 6.6 the rhtB gene which imparts homoserine resistance,6.7 the mqo gene which codes for malate:quinone oxidoreductase, 6.8 therhtC gene which imparts threonine resistance, 6.9 the thrE gene whichcodes for the threonine export protein, 6.10 the gdhA gene which codesfor glutamate dehydrogenase, 6.11 the hns gene which codes for theDNA-binding protein HLP-II, 6.12 the pgm gene which codes forphosphoglucomutase, 6.13 the fba gene which codes for fructosebiphosphate aldolase, 6.14 the ptsH gene which codes for thephosphohistidine protein hexose phosphotransferase, 6.15 the ptsI genewhich codes for enzyme I of the phosphotransferase system, 6.16 the crrgene which codes for the glucose-specific IIA component, 6.17 the ptsGgene which codes for the glucose-specific IIBC component, 6.18 the lrpgene which codes for the regulator of the leucine regulon, 6.19 the mopBgene which codes for 10 Kd chaperone, 6.20 the ahpc gene which codes forthe small sub-unit of alkyl hydroperoxide reductase, 6.21 the ahpF genewhich codes for the large sub-unit of alkyl hydroperoxide reductase,6.22 the cysK gene which codes for cysteine synthase A, 6.23 the cysBgene which codes for the regulator of the cys regulon, 6.24 the cysJgene which codes for the flavoprotein of NADPH sulfite reductase, 6.25the cysI gene which codes for the haemoprotein of NADPH sulfitereductase and 6.26 the cysH gene which codes for adenylyl sulfatereductase, is or are enhanced, in particular over-expressed, arefermented.
 7. A process as claimed in claim 1, wherein, for thepreparation of L-amino acids, microorganisms of the Enterobacteriaceaefamily in which in addition at the same time one or more of the geneschosen from the group consisting of: 7.1 the tdh gene which codes forthreonine dehydrogenase, 7.2 the mdh gene which codes for malatedehydrogenase, 7.3 the gene product of the open reading frame (orf)yjfA, 7.4 the gene product of the open reading frame (orf) ytfP, 7.5 thepckA gene which codes for phosphoenol pyruvate carboxykinase 7.6 thepoxB gene which codes for pyruvate oxidase 7.7 the aceA gene which codesfor isocitrate lyase, 7.8 the dgsA gene which codes for the DgsAregulator of the phosphotransferase system, 7.9 the fruR gene whichcodes for the fructose repressor, 7.10 the rpoS gene which codes for thesigma³⁸ factor, 7.11 the aspA gene which codes for aspartate ammoniumlyase, 7.12 the aceB gene which codes for malate synthase A and 7.13 theugpB gene which codes for the periplasmic binding protein of thesn-glycerol 3-phosphate transport system is or are attenuated, inparticular eliminated or reduced in expression, are fermented.